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In JoVE (1)
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Articles by Helit Nabel-Rosen in JoVE
JoVE General
दो फोटॉन आधारित लाइव Zebrafish भ्रूण में photoactivation
Molecular Cell Biology, Weizmann Institute of Science
माइक्रोस्कोपी multiphoton गहरी ऑप्टिकल पैठ और कम phototoxicity के साथ कम ऊर्जा photons के नियंत्रण की अनुमति देता है. हम zebrafish भ्रूण में रहते सेल लेबलिंग के लिए इस प्रौद्योगिकी के उपयोग का वर्णन. यह प्रोटोकॉल विभिन्न प्रकाश उत्तरदायी अणुओं की तस्वीर शामिल होने के लिए आसानी से अनुकूलित किया जा सकता है.
Other articles by Helit Nabel-Rosen on PubMed
Cell Divisions in the Drosophila Embryonic Mesoderm Are Repressed Via Posttranscriptional Regulation of String/cdc25 by HOW
Cell-cycle progression is tightly regulated during embryonic development. In the Drosophila early embryo, the levels of String/Cdc25 define the precise timing and sites of cell divisions. However, cell-cycle progression is arrested in the mesoderm of gastrulating embryos despite a positive transcriptional string/cdc25 activation provided by the mesoderm-specific action of Twist. Whereas String/Cdc25 is negatively regulated by Tribbles in the mesoderm at these embryonic stages, the factor(s) controlling string/cdc25 mRNA levels has yet to be elucidated.
Specification of Hypothalamic Neurons by Dual Regulation of the Homeodomain Protein Orthopedia
In the developing hypothalamus, a variety of neurons are generated adjacent to each other in a highly coordinated, but poorly understood process. A critical question that remains unanswered is how coordinated development of multiple neuronal types is achieved in this relatively narrow anatomical region. We focus on dopaminergic (DA) and oxytocinergic (OT) neurons as a paradigm for development of two prominent hypothalamic cell types. We report that the development of DA and OT-like neurons in the zebrafish is orchestrated by two novel pathways that regulate the expression of the homeodomain-containing protein Orthopedia (Otp), a key determinant of hypothalamic neural differentiation. Genetic analysis showed that the G-protein-coupled receptor PAC1 and the zinc finger-containing transcription factor Fezl act upstream to Otp. In vivo and in vitro experiments demonstrated that Fezl and PAC1 regulate Otp at the transcriptional and the post-transcriptional levels, respectively. Our data reveal a new genetic network controlling the specification of hypothalamic neurons in vertebrates, and places Otp as a critical determinant underlying Fezl- and PAC1-mediated differentiation.
Dopaminergic Neuronal Cluster Size is Determined During Early Forebrain Patterning
We have explored the effects of robust neural plate patterning signals, such as canonical Wnt, on the differentiation and configuration of neuronal subtypes in the zebrafish diencephalon at single-cell resolution. Surprisingly, perturbation of Wnt signaling did not have an overall effect on the specification of diencephalic fates, but selectively affected the number of dopaminergic (DA) neurons. We identified the DA progenitor zone in the diencephalic anlage of the neural plate using a two-photon-based uncaging method and showed that the number of non-DA neurons derived from this progenitor zone is not altered by Wnt attenuation. Using birthdating analysis, we determined the timing of the last cell division of DA progenitors and revealed that the change in DA cell number following Wnt inhibition is not due to changes in cell cycle exit kinetics. Conditional inhibition of Wnt and of cell proliferation demonstrated that Wnt restricts the number of DA progenitors during a window of plasticity, which occurs at primary neurogenesis. Finally, we demonstrated that Wnt8b is a modulator of DA cell number that acts through the Fz8a (Fzd8a) receptor and its downstream effector Lef1, and which requires the activity of the Fezl (Fezf2) transcription factor for this process. Our data show that the differential response of distinct neuronal populations to the Wnt signal is not a simple interpretation of their relative anteroposterior position. This study also shows, for the first time, that diencephalic DA population size is modulated inside the neural plate much earlier than expected, concomitant with Wnt-mediated regional patterning events.
High Resolution Fate Map of the Zebrafish Diencephalon
The diencephalon acts as an interactive site between the sensory, central, and endocrine systems and is one of the most elaborate structures in the vertebrate brain. To better understand the embryonic development and morphogenesis of the diencephalon, we developed an improved photoactivation (uncaging)-based lineage tracing strategy. To determine the exact position of a given diencephalic progenitor domain, we used a transgenic line driving green fluorescent protein (GFP) in cells expressing the proneural protein, Neurogenin1 (Neurog1), which was used as a visible neural plate landmark. This approach facilitated precise labeling of defined groups of cells in the prospective diencephalon of the zebrafish neural plate. In this manner, we labeled multiple overlapping areas of the diencephalon, thereby ensuring both accuracy and reproducibility of our lineage tracing regardless of the dynamic changes of the developing neural plate. We present a fate map of the zebrafish diencephalon at a higher spatial resolution than previously described.
